The mTOR inhibitor CCI-779 induces apoptosis and inhibits growth in preclinical models of primary adult human ALL

Abstract

Acute lymphoblastic leukemia (ALL) in adult patients is often resistant to current therapy, making the development of novel therapeutic agents paramount. We investigated whether mTOR inhibitors (MTIs), a class of signal transduction inhibitors, would be effective in primary human ALL. Lymphoblasts from adult patients with precursor B ALL were cultured on bone marrow stroma and were treated with CCI-779, a second generation MTI. Treated cells showed a dramatic decrease in cell proliferation and an increase in apoptotic cells, compared to untreated cells. We also assessed the effect of CCI-779 in a NOD/SCID xenograft model. We treated a total of 68 mice generated from the same patient samples with CCI-779 after establishment of disease. Animals treated with CCI-779 showed a decrease in peripheral-blood blasts and in splenomegaly. In dramatic contrast, untreated animals continued to show expansion of human ALL. We performed immunoblots to validate the inhibition of the mTOR signaling intermediate phospho-S6 in human ALL, finding down-regulation of this target in xenografted human ALL exposed to CCI-779. We conclude that MTIs can inhibit the growth of adult human ALL and deserve close examination as therapeutic agents against a disease that is often not curable with current therapy.

Figures

Figure 1.

CCI-779 induces apoptosis in a short-term human ALL culture system. A total of 106 lymphoblasts from patients with adult human ALL were aliquoted onto a bone marrow stromal cell layer. All cells were maintained in culture for 24 hours and were either untreated (control) or treated with CCI-779 (100 ng/mL) for 12 or 24 hours. The x-axis depicts patient sample identification, and the y-axis depicts the percentage of apoptotic (annexin V-positive)/CD19-positive cells.

Figure 2.

Establishment of xenografts. NOD/SCID mice were irradiated with 275 cGY and were injected with 107 lymphoblasts obtained from patients with pre-B ALL. Xenografts were established from 78% of patient samples used as assessed by flow cytometry for ALL. Top panel shows flow cytometric analysis of CD45 and CD19 staining of peripheral blood from a control mouse (A, 0.1%) and a xenografted mouse (D, 32%). Splenocytes from this mouse were 65% human ALL (B), and bone marrow was 94% human ALL (C). The majority of xenografted animals had more than 90% replacement of bone marrow and more than 60% replacement of spleen with human ALL. Also pictured are photomicrographs of Wright-Giemsa-stained peripheral blood (E) with human lymphoblast (arrow) and murine granulocyte (shown) and a cytospin prepared from bone marrow nucleated cells showing a monomorphic population of lymphoblasts (F). Images were captured using a Zeiss Axiovert 40C light microscope (Carl Zeiss, Thornwood, NJ) equipped with an apochromatic 40 ×/0.60 NA objective lens and a Nikon 995 camera (Nikon, Melville, NY).

Figure 3.

CCI-779 is efficacious in ALL xenografts. NOD/SCID mice were xenografted with human ALL from patient samples. After establishment of disease, defined as more than 5% blasts detected in peripheral blood, mice were randomized to treatment with 5 to 10 mg/kg/d of CCI-779 versus vehicle control. Disease was evaluated at weekly intervals by FACS analysis of peripheral blood, detecting human CD19+ and CD45+ cells. Graph depicts mean absolute blast counts (WBC × % blasts by FACS analysis) from mice generated from the 4 patient samples at weekly intervals, demonstrating statistically significant difference (P < .05) in all samples. Error bars depict standard error of the mean (SEM).

Figure 4.

Response of splenic disease to CCI-779 treatment. Xenografted mice with established disease (> 5% peripheral blasts) were randomized to treatment with 5 to 10 mg/kg/d of CCI-779 versus control (vehicle). Lymphoblasts in spleen in treated and untreated mice were compared at time of death. Graph represents number of blasts in millions (× 106), demonstrating statistically significant difference (P < .05) in 3 of 4 patient samples. Splenic blast counts were calculated by multiplying total number of cells in spleens by percent of anti-human CD19+ and anti-human CD45+ cells as determined by FACS analysis. Error bars depict SEM.

Figure 5.

Treatment with MTIs results in hypophosphorylated S6 in ALL. Splenocytes were harvested from mice treated with CCI-779 or control (vehicle). Immunoblot of phospho-S6 (ser235/236) of control and CCI-779 treated (top bands), total S6 (middle bands), and β-tubulin (bottom band; loading control) from 4 patient samples is depicted. We found a correlation between clinical response and biochemical response to CCI-779 in all samples. ALL cells from CCI-779-treated mice had 48% to more than 95% down-regulation of phospho-S6 compared to blasts from control mice.